Paul.xu at 19:00, 3 February 2021

2021-02-03T19:00:45Z

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== Protocol 1: Gibson Assembly for Cloning PCR Fragment into a Vector ==

1) Prepare 6 ml of '''5X ISO Buffer '''in a 15 ml falcon tube as follows:

3 ml 1 M Tris-HCl pH 7.5

+ 150µl 2 M MgCl2

+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)

+ 300µl 1 M DTT

+ 1.5 g PEG-8000

+ 300µl 100 mM NAD

+ dH20 to 6 ml

Store at -20 oC in 320 µl aliquots.

2) Prepare '''0.5  ml''' of '''3.2 X Gibson Cloning master mix''' as follows:

320 µl 5X ISO Buffer

+ 0.64 µl 10 U/µl T5 exonuclease*

+ 20 µl 2 U/µl Phusion polymerase

+ 160 µl 40 U/µl Taq ligase

'''DO NOT ADD WATER'''

Store at -20 oC in '''5 µl '''aliquots.This is 3.2 X mix. This mix allows you to add sample and water to 16 µl

This is optimized for 20-150 bp sequence homology overlaps

3) Thaw a '''5 µl '''aliquot of 3.2X Gibson assembly master mix, and keep on ice until use.

4) Measure the DNA concentration (ng/µl) of each assembly piece.

5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces in '''11µl''' (e.g., elute DNA from spin column with 11 µl water after gel purification) to the thawed '''5µl''' 3.2X master mix in a '''16 µl''' total volume assembly reaction mixture as follows:

linearized vector backbone (100 ng) + each additional assembly piece (to equimolar with backbone)==11  µl

+ 5 µl 3.2 X Gibson Cloning assembly master mix

6) Incubate the assembly reaction at '''50 oC '''for 15 ~ '''60 minutes''', and then place on ice.

7) Transform 5 ~15 µl of the assembly reaction into 100µl of competent E. coli and/or run an agarose gel to check for successful assembly.

== Gibson Assembly Protocol for Joining Two or More PCR Fragments (Gene Synthsis) ==

1) Prepare 6 ml of '''5X ISO Buffer''' in a 15 ml falcon tube as follows:

3 ml '''1 M Tris-HCl pH 7.5'''

+ 150µl 2 M MgCl2

+ 240 µl 100 mM dNTP mix (25 mM each: dGTP, dCTP, dATP, dTTP)

+ 300µl 1 M DTT

+ 1.5 g PEG-8000

+ 300µl 100 mM '''NAD'''

+ dH20 to 6 ml

Store at -20 oC in 320µl aliquots.

2) Prepare 0.5 ml of '''Gibson assembly master mix '''as follows:

320µl 5X ISO Buffer

+ 0.64 µl 10 U/µl T5 exonuclease*

+ 20 µl 2 U/µl Phusion polymerase

+ 160 µl 40 U/µl Taq ligase

<strike>+ dH20 to 1.2 ml</strike>

Store at -20 oC in <strike>15</strike> 5 µl aliquots.

This is optimized for 20-150 bp sequence homology overlaps

3) Thaw a <strike>15</strike> 5 µl aliquot of the Gibson assembly master mix, and keep on ice until use.

4) Add purified PCR fragments, or unpurified PCR products

5) Add water to <strike>20</strike> 16 µl

6) Incubate at 50 oC water bath for 1 hour.

7). Run agarose gel and cut out right size band, use Qiagen agarose gel extraction kit to purify DNA

8). Digest with enzyme and clone into a digested vector.

<strike>5) Add 100 ng of the linearized vector backbone and equimolar amounts of the other assembly pieces to the thawed 15µl master mix in a 20 µl total volume assembly reaction mixture as follows:</strike>

<strike>linearized vector backbone (100 ng)</strike>

<strike>+ each additional assembly piece (to equimolar with backbone)</strike>

<strike>+ 15µl Gibson assembly master mix</strike>

<strike>+ dH20 to 20µl</strike>

<strike>6) Incubate the assembly reaction at 50 oC for 15 ~ 60 minutes, and then place on ice.</strike>

<strike>7) Transform 5 ~20 µl of the assembly reaction into 100µl of competent E. coli and/or run an agarose gel to check for successful assembly.</strike>

<strike>A major limitation (especially in high GC rich templates) to Gibson assembly is that the termini of the DNA sequence fragments to be assembled should not have stable single stranded DNA secondary structure, such as a hairpin or a stem loop, or repeated sequences, as this would directly compete with the required single-stranded annealing/priming of neighboring assembly fragments.</strike>

== Protocol 3 ==

==== Gibson Assembly ====

#Prepare the Assembly Mix on ice
#Incubate for one hour at 50°C
#Optional gel purification
#Transform

{|
|-
! Assembly Mix
! Volume (μl)
|-
| Gibson Master Mix
| 15
|-
| Total DNA
| 5
|-
! Total
| 20
|}

For instance, if you are joining DNA fragments, you would use about 1.25μl of each.

{|
|-
! Gibson Master Mix
! Volume (μl)
|-
| Taq ligase (40u/µl)
| 50
|-
| 5x isothermal buffer
| 100
|-
| T5 exonuclease (1u/µl)
| 2
|-
| Phusion polymerase (2u/µl)
| 6.25
|-
| Nuclease-free water
| 216.75
|-
! Total
| 375
|}

Whilst the mixture can be refrozen, for best performance you should aliquot it into 25x15μl.

{|
|-
! 5x isothermal buffer
! Volume (µl)
|-
| 25% PEG-8000
| 0.75g
|-
| 500 mM Tris-HCl, pH 7.5
| 1500
|-
| 50mM MgCl2
| 75
|-
| 50mM DTT
| 150
|-
| 1mM dATP
| 30
|-
| 1mM dTTP
| 30
|-
| 1mM dCTP
| 30
|-
| 1mM dGTP
| 30
|-
| 5mM NAD
| 300
|-
| Nuclease-free water
| ...
|-
! Total
| 3000
|}

Use nuclease-free water to make up to 3000μl. In this case, very little water is needed as the volume is 2145μl + 0.75g.

DNA molecules overlapping by larger than 150 bp, prepare the ssembly mixture by using 3.2 μl of 10 U/ μl T5 exo.

== Protocol 3 ==

'''5x isothermal assembly reaction buffer (assemble on ice):'''

Added: 3 mL 1M Tris-HCl pH 7.5 150 uL 2M MgCl2 or 300 uL 1M MgCl2

60 uL 100 mM dGTP 60 uL 100 mM dCTP 60 uL 100 mM dTTP 60 uL 100 mM dATP

or 600 uL 10 mM dNTP mix

 300 uL 1M DTT 1.5 g PEG-8000 300 uL 100 mM or  NAD 20 mg NAD ddH2O to 6 mL

Prepare 320 uL aliquots (18) and freeze all but one.

Label these “5X isotherm buffer”

To the one remaining (320 uL), add: 1.2 uL T5 Exonuclease 20 uL Phusion polymerase (NOT HOTSTART) 160 uL Taq ligase 700 uL ddH2O

Prepare 15 uL aliquots (~80) on ice in PCR tubes and store at -20C. These should be good for up to a year.

== Enhancement ==

5x Isothermal Reaction Mix was created by combining 3 ml 1 M Tris-HCl, pH = 7.5 (ThermoFisher Scientific 15567027), 300 µl 1 M MgCl2 (Millipore Sigma M1028), 600 µl 10 mM dNTP mix (Goldbio D-900-1), 46.3 mg DTT (Goldbio DTT10), 20 mg NAD (Millipore Sigma N0632), 1.5 g PEG  8000 (Millipore Sigma 89510), and brought to 6 ml with UltraPure water (ThermoFisher Scientific 10977015). 320 µl aliquots were stored at -80C. To create a 1.33x Gibson Assembly mix, 40 units Phusion High-Fidelity DNA Polymerase (NEB M0530), 12 units T5 exonuclease (NEB M0363), and 6,400 units Tth DNA Ligase (homemade, supplementary protocol 1) were added to 320 ul of 5x Isothermal Reaction Mix. For the enhanced formulation, '''10 ul ET SSB (500 µg/ml, NEB M2401S)'''was also added. UltraPure water (ThermoFisher Scientific 10977015) was added to bring the final volume of each to 1.2 ml. Gibson Assembly reaction mixes were dispensed into single use aliquots and stored at -20C. 

Assembly Reactions All reactions included each insert fragment at a 6-fold molar excess to the vector fragment. Each 10 µl assembly reaction included 4 fmol of vector along with 24 fmol of each insert fragment and 0.4 fmol of an intact supercoiled kanamycin-resistant plasmid (as a transformation control). For both the original and enhanced Gibson Assembly formulations, 2.5 µl of this DNA mixture was added to 7.5 µl of 1.33x master mix on ice. For the NEBuilder® HiFi reactions, 2.5 µl of this DNA mixture and 2.5 µl of UltraPure water was added to 5 µl of NEBuilder® HiFi DNA Assembly Master Mix (NEB E2621) on ice. After mixing, the reactions were moved directly to 50C for one hour and then placed back on ice. Both the 2-insert and 6-insert assemblies were run in triplicate for each reaction formulation.

Transformation 15 µl of UltraPure water was added to each assembly reaction on ice and 2.5 µl of these diluted reactions were then transformed into 50 µl chemically competent DH5α cells (prepared in house, Zymo T3001) on ice. Following a five minute incubation on ice, 450 µl of pre-warmed SOC outgrowth medium (NEB B9020S) was added and the transformation was incubated at 37C with shaking (250 RPM) for one hour. 200 µl of each transformation was then plated on an LB-agar plate with carbenicillin (100 µg/ml) and another 200 µl was plated on an LB-agar plate with kanamycin (50 µg/ml). Plates were incubated overnight at 37C Compared to the original Gibson Assembly formulation, this enhanced formulation exhibited markedly increased accuracy for the 6-insert assembly (>80% compared to ~20%, p = 3e-5), and greatly increased efficiency in both 2-insert and 6-insert assemblies (>12-fold increase, p < 0.05). Compared to the NEBuilder® HiFi DNA Assembly Master Mix (NEB E2621), the enhanced formulation showed a trend toward higher assembly efficiency, but this trend failed to reach statistical significance. However, the enhanced formulation showed significantly increased accuracy in the 6-insert assembly (>80% compared to ~45%, p = 0.00024). These results clearly demonstrate that the addition of ET SSB can increase the efficiency and accuracy of Gibson Assembly, especially as the number of fragments being assembled increases. While the exact mechanism has yet to be conclusively elucidated, it is possible that this ssDNA binding protein may facilitate specific hybridization and protect ssDNA overhangs from endonuclease attack.4, 6 The simplicity of this modification makes it easy to use in place of the original formulation without any other modifications. Furthermore, by making this mix in-house with homemade Thermus thermophilus DNA Ligase, this mix can be made for less than $10 per milliliter, making it accessible in a wide variety of research settings. 

Homemade Assembly Mixes Both the original and enhanced Gibson Assembly mixes were based on recipes from OpenWetWare. 9 Briefly, a 5x Isothermal Reaction Mix was created by combining 3 ml 1 M Tris-HCl, pH = 7.5 (ThermoFisher Scientific 15567027), 300 µl 1 M MgCl2 (Millipore Sigma M1028), 600 µl 10 mM dNTP mix (Goldbio D-900-1), 46.3 mg DTT (Goldbio DTT10), 20 mg NAD (Millipore Sigma N0632), 1.5 g PEG 8000 (Millipore Sigma 89510), and brought to 6 ml with UltraPure water (ThermoFisher Scientific 10977015). 320 µl aliquots were stored at -80C. To create a 1.33x Gibson Assembly mix, 40 units Phusion High-Fidelity DNA Polymerase (NEB M0530), 12 units T5 exonuclease (NEB M0363), and 6,400 units Tth DNA Ligase (homemade, supplementary protocol 1) were added to 320 ul of 5x Isothermal Reaction Mix. For the enhanced formulation, 10 ul ET SSB (500 µg/ml, NEB M2401S) was also added. UltraPure water (ThermoFisher Scientific 10977015) was added to bring the final volume of each to 1.2 ml. Gibson Assembly reaction mixes were dispensed into single use aliquots and stored at -20C. 

== Summary ==

Gibson Chew Back and Anneal Assembly (Gibson CBA) is a quick and easy method to construct plasmids without using restriction enzymes. In this method, DNA fragments to be assembled are PCR amplified with 40 bp of overlap to the adjacent sequence (only one DNA fragment per interface needs to have an overlap extension into the adjacent fragment). These fragments are then mixed in a single pot with a single strand exonuclease to generate sticky ends and allowed to anneal before being repaired by a polymerase and a ligase. In theory, many fragments can be assembled simultaneously (5, 4 inserts + backbone, have been documented in the [[Prather:Gibson_CBA#References|initial report]]) into a single product of up to ~100 kb+. The method has several thermocycled variants of higher efficiency but the isothermal protocol offers almost comparable yields with greater simplicity.

== Materials ==

*Master Mix
**Store in -20C Freezer

Prather Recipe '''5x Isothermal Reaction Mix'''

:3 ml 1 M Tris-Hcl (pH 7.5) 
:300 μL 1 M MgCl2 
:60 μL 100 mM dGTP 
:60 μL 100 mM dATP 
:60 μL 100 mM dTTP 
:60 μL 100 mM dCTP 
:300 μL 1 M DTT 
:1.5 g PEG-8000 
:300 μL 100 mM NAD 
:<ins>balance ddH2O</ins> 
:6 ml Total

'''Assembly Master Mix'''

:320 μL 5X Isothermal Master Mix
:0.64 μL 10 U/μL T5 exonuclease
:20 μL 2 U/μL Phusion DNA Pol
:160 μL 40 U/μL Taq DNA Ligase
:<ins>860 μL ddH2O</ins>
:1.2 ml Total

[http://www.cambridgeigem.org/RFC57.pdf RFC57] Recipe '''5x isothermal reaction buffer:'''

*25% PEG-8000
*500 mM Tris-HCl pH 7.5
*50 mM MgCl2,
*50mM DTT
*5mM NAD
*1mM each of the four dNTPs

'''1.33x Gibson Master Mix:'''

*Taq ligase (40u/ul): 50 ul
*5x isothermal buffer: 100 ul
*T5 exonuclease (1u/ul): 2 ul
*Phusion polymerase (2u/ul): 6.25 ul
*Nuclease-free water: 216.75 ul

== Gibson Method, aka VLIC, aka one pot isothermal assembly ==

Original protocol from: [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html this paper], doi:10.1038/nmeth.1318

#PCR up your fragment of choice and gel purify
#Thaw a 15 μl assembly mixture aliquot and keep on ice until ready to be used.
#Add 5 μl of DNA to be assembled to the master mixture. The DNA should be in equimolar amounts. Use 10-100 ng of each ~6 kb DNA fragment. For larger DNA segments, increasingly proportionate amounts of DNA should be added (e.g. 250 ng of each 150 kb DNA segment).
#Incubate at 50 °C for 15 to 60 min (60 min is optimal).
#Transform as usual

== Notes ==

*Ideally you have an overlap of 40 bp
*Misc: - rxn product is salty: can be a problem for electrocompetent cells. Rob Egbert dilutes the competent cells when transforming with electroporation. Not so much a problem when using chemically competent cells. - Electrocompetence is ~ 1000x more effective so is tempting.
*When preparing the isothermal reaction mix, add the PEG slowly to liquid. If added too quickly it will form a plug which will make mixing difficult (KS)
*The initial paper suggests that 10 - 100 ng of total DNA be used for assemblies. I've gone as high as 170 ng without any ill effects. (KS)
*Have successfully used for a two way and three way ligation (KS)
*There is a potential for mutations at the DNA boundaries which has yet to be quantified. Paper suggests 1 every 50 assemblies or so. Of the two initial assemblies I made, one had a missense mutation so sequence to verify interfaces or leave spacers (~ 50 bp or so) at the interfaces to 'absorb' these errors (KS)
*I have used PCRs as is (with PCR cleanup only) and gel extracted DNA in my assemblies. PCR cleanup gives more colonies (more DNA, better quality (no agarose/QG contamination)) but also has more false positives (PCR template plasmid). False positives may be alleviated by DpnI treatment if gel extraction is not used but I haven't tested this yet (KS).
*I once inadvertently designed my primers with 20 bp homology and a 20 bp spacer from the adjacent fragment and still got accurate plasmids. Possible to use less overlap if desired (KS)

== Specific Protocols ==

*[[Prather:Gibson_CBA]]
*[[User:Andrew_Perry/Notebook/bbGibson/Gibson_assembly_protocol]]
*[[Springer_Lab:_Isothermal_Assembly]]
*[[Janet_B._Matsen:Guide_to_Gibson_Assembly|Guide to Gibson Assembly]]

== Protocol 4 ==

MATERIALS

beta-Nicotinamide adenine dinucleotide (NAD+) - 0.2 ml, New England Biolabs Catalog #B9007S

Q5 High-Fidelity DNA Polymerase - 100 units , New England BiolabsCatalog #M0491S

Taq DNA Ligase - 2,000 units, New England BiolabsCatalog #M0208S

T5 Exonuclease - 5,000 units, New England BiolabsCatalog #M0363L

PEG-8000 

DTT (Dithiothreitol) (> 99% pure) Protease free,  Gold Biotechnology Catalog #DTT

Deoxynucleotide Solution Set - 25 umol of each, New England Biolabs Catalog #N0446S

Recipe for 4 mL:

{|
|-
| colspan="1" rowspan="1" | Component
| colspan="1" rowspan="1" | Molarity / Concentration
| colspan="1" rowspan="1" | Amount
| colspan="1" rowspan="1" | Final concentration
|-
| colspan="1" rowspan="1" | Tris-HCl, pH 7.5
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 2 mL
| colspan="1" rowspan="1" | 500 mM
|-
| colspan="1" rowspan="1" | MgCl2
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 50 mM
|-
| colspan="1" rowspan="1" | dATP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dCTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dGTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dTTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | DTT
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 50 mM
|-
| colspan="1" rowspan="1" | PEG-8000
| colspan="1" rowspan="1" | -
| colspan="1" rowspan="1" | 1 g
| colspan="1" rowspan="1" | 25 %
|-
| colspan="1" rowspan="1" | NAD+
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 5 mM
|-
| colspan="1" rowspan="1" | H2O
| colspan="1" rowspan="1" | -
| colspan="1" rowspan="1" | to final volume of 4 mL
| colspan="1" rowspan="1" | -
|}

Mix dNTPs, NAD+, Tris-HCl, MgCl2 and DTT.

Slowly add PEG-8000 to mixture and mix well, until completely dissolved. Add H2O to a final volume of 4 mL.

Prepare aliquots of the 5x isothermal buffer as required, e.g. 100 µL. Store at -20 ˚C. Preparation of 1,33x Assembly Mastermix

Recipe for 25 x 15 µL aliquots:

{|
|-
| colspan="1" rowspan="1" | ''' Component'''
| colspan="1" rowspan="1" | ''' Concentration'''
| colspan="1" rowspan="1" | ''' Amount'''
| colspan="1" rowspan="1" | ''' Final concentration (after adding DNA)'''
|-
| colspan="1" rowspan="1" |  5x isothermal rxn buffer
| colspan="1" rowspan="1" |  5x
| colspan="1" rowspan="1" |  100 µL
| colspan="1" rowspan="1" |  1x
|-
| colspan="1" rowspan="1" |  Taq DNA Ligase
| colspan="1" rowspan="1" |  40 U/µL
| colspan="1" rowspan="1" |  50 µL
| colspan="1" rowspan="1" |  4 U/µL 
|-
| colspan="1" rowspan="1" |  T5 Exonuclease
| colspan="1" rowspan="1" |  1 U/µL
| colspan="1" rowspan="1" |  2 µL
| colspan="1" rowspan="1" |  4 U/mL
|-
| colspan="1" rowspan="1" |  Q5 Hi-Fi DNA Polymerase
| colspan="1" rowspan="1" |  2 U/µL
| colspan="1" rowspan="1" |  6.25 µL
| colspan="1" rowspan="1" |  25 U/mL
|-
| colspan="1" rowspan="1" |  H2O
| colspan="1" rowspan="1" |
| colspan="1" rowspan="1" |  216.75
| colspan="1" rowspan="1" |
|-
| colspan="1" rowspan="1" |  '''Total volume'''
| colspan="1" rowspan="1" |
| colspan="1" rowspan="1" | '''375 µL'''
| colspan="1" rowspan="1" |
|}

Preparation of 1.33x Assembly Mastermix

Work on ice. Mix H2O and 5x buffer, then add enzymes.

Prepare 25 x 15 µL aliquots in PCR tubes. Store at -20 ˚C.

These aliquots are concentrated 1.33 x - add your DNA in a volume of 5 µL to a final volume/concentration of 20 µL / 1x. Gibson assembly

After addition of DNA, incubate Gibson assembly mix at 50 ˚C for 45 min.
[[Category:Protocols]]

Gibson Assembly - Revision history

Paul.xu at 19:00, 3 February 2021